We have previously found that docosahexaenoic acid (DHA, 22:6n-3), a highly polyunsaturated n-3 fatty acid, promotes the accumulation of phosphatidylserine (PS) and prevents apoptotic neuronal cell death. We have also demonstrated that n-3 fatty acid deficiency or chronic ethanol exposure markedly decreased the PS content specifically in neuronal cells where DHA is highly enriched. Under n-3 fatty acid deficiency, DHA is replaced by docosapentaenoic acid (DPA, 22:5n-6). We have established that DPA is not as effective as DHA in accumulating PS or preventing apoptotic cell death, thus adversely affecting neuronal survival under n-3 deficiency conditions. Similarly, long term ethanol exposure during prenatal and developmental period depletes PS from neuronal membranes and promotes apoptotic neuronal cell death. During this period, we have investigated the biochemical and molecular mechanisms underlying these effects. To elucidate the biochemical mechanism for the PS modulation by the DHA status and ethanol, the effect of n-3 deficiency and ethanol exposure on PS biosynthetic activity was examined. Rats were fed with n-3 deficient diet (15% 18:2n-6 and 0.02% 18:3n-3) or n-3 fatty acid adequate diet (15% 18:2n-6 and 3% 18:3n-3) during pregnancy (E2-E18) and lactation period. To study the effect of ethanol, rats were fed with ethanol mixed in an AIN-93G based diet from day 11 of gestation during pregnancy and throughout the lactation period. Microsomal fractions obtained from the brain cortex of the off spring were incubated with deuterium labeled PS or PE species in vitro and the production of labeled PS species was monitored using reversed phase liquid chromatography/ electrospray ionization mass spectrometry for PSS1 or PSS2 activity, respectively. We found that both PS synthetic activity by both PSS1 and PSS2 was highest for the 18:0,22:6-PS species. Feeding rats with ethanol significantly decreased both PSS1 and PSS2 biosynthetic activity, resulting in the reduction of PS levels in neuronal tissues in agreement with our previous findings. N-3 fatty acid deficiency, however, did not affect the PS synthetic activity itself from exogenous substrates, suggesting that the loss of PS is due to the loss of the 18:0, 22:6 species, the favorite substrate for PS synthesis. To investigate the signaling mechanisms supporting the PS-dependent protective effect of DHA on neuronal survival, we examined the IGF stimulated growth factor receptor signaling. When the PI3-kinase/Akt pathway was evaluated using the green fluorescent tagged PH domain of Akt (GFP-PH-Akt), the translocation of GFP-PH-Akt in response to IGF was significantly faster in DHA enriched cells in comparison to non-enriched control cells. When cells were enriched with DHA in serine free condition, PS accumulation was inhibited and the accelerated translocation of the PH domain was no longer observed. Akt phosphorylation also followed a similar time course with faster phosphorylation with DHA enrichment. The cells enriched with DPA also showed faster translocation in comparison to non-enriched control, but at a slower rate than DHA enriched cells. These data are consistent with our previous finding that DHA is more efficient in promoting cell survival in comparison to DPA, indicating that Akt-translocation is a target for the protective effect of DHA. To extend our in vitro finding that DHA uniquely promotes hippocampal differentiation, we have evaluated the effect of in vivo modulation of the DHA status on neurite outgrowth. E-18 hippocampi obtained from the fetuses of n-3 fatty acid deficient rats contained 42.4% less DHA content in comparison to those obtained from the n-3 fatty acid adequate fetus. N-3 fatty acid deficient hippocampal cultures showed shorter total neurite lengths after 6 days in vitro culture in comparison to the n-3 fatty acid adequate cultures. Supplementation of the n-3 fatty acid deficient cultures with 1.5 microM DHA increased the total neurite length to a level similar to the n-3 fatty acid adequate group. These data are in agreement with our previous finding that DHA has a unique effect on promoting neurite outgrowth and indicate that provision of DHA during the developmental period is important for neuronal differentiation. As a part of our continuing efforts to investigate protein conformation, rhodopsin conformational changes during visual signal transduction were examined during this period using intra-molecular cross linking and mass spectrometric analysis. While dark-adapted rhodopsin showed cross-linking between the C-1 loop and the C-terminus, cross-linking between C-2 and C-4 loops were observed after light exposure, suggesting that the cytoplasmic loops covered by the C-terminal tail under the dark-adapted condition become exposed due to the light exposure.